USPTO Patents Application 10561839

BEST AVAILABLE COPY

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(19) World Intellectual Property Organization

International Bureau

(43) International Publication Date
15 August 2002 (15.08.2002)

PCT

(10) International Publication Number

WO 02/063034 Al

(51) International Patent Classification 7 :

C12M 1/34

C12Q 1/24,

(21) International Application Number: PCT/US02/02660

(22) International Filing Date: 31 January 2002 (31.01.2002)

(25) Filing Language: English

(26) Publication Language: English

(30) Priority Data:

09/773,583

2 February 2001 (02.02.2001) US

(71) Applicant (for all designated States except US): MEDIS
EL LTD. [IL/IL]; 14 Shebazi St., 56400 Yehud (IL).

(71) Applicant (for TJ only): FRIEDMAN, Mark, M.

[US/IL]; 1 Alharizi St., 43406 Raa nana (IL).

(72) Inventors; and

(75) Inventors/Applicants (for US only): HUBERMAN,

Tamir [IL/IL]; 3 David Kind Street, 76400 Rehovot (IL).
SARIN, Alexander [IL/IL]; 23/15 Zeelim St., 93896
Jerusalem (IL). DANGOUR, Doron [IL/IL]; 3/13 Dan St.,
71477 Lod (IL).

(74) Common Representative: FRIEDMAN, Mark, M.; do

Discovery Dispatch, 9003 Florin Way, Upper Marlboro,
MD 20772 (US).

(81) Designated States (national): AE, AG, AL, AM, AT, AU,
AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH t
GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
MX, MZ, NO, NZ, OM, PH, PL, PT, RO, RU, SD, SE, SG,
SI, SK, SL, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ,
VN, YU, ZA, ZM, ZW.

(84) Designated States (regional): ARIPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),

[Continued on next page]

(54) Title: AN IMPROVED SYSTEM AND METHOD FOR COLLECTING DATA FROM INDIVIDUAL CELLS

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(57) Abstract: An automated system (70) and method for
loading individual cells into individual discrete locations (77).
The system includes a cell carrier grid (5), a cell carrier grid
holder (71), a vacuum source (74), a liquid reservoir (75) and
a loading device (76) facilitating communication between the
above components. Application of vacuum via a port causes
cells to move into discrete locations. The method includes the
steps of placing the grid holder into a loading device, automat-
ically filling a space in the grid holder with a liquid, automati-
cally adding to an upper surface of the grid and automatically
applying a force to the cells in so that individual cells enter
at least some of the individual discrete locations. Further dis-
closed is an automated system for collection of data from the
cells further including an electro-optical scanner (25) capa-
ble of illuminating the cells and collecting at least a portion
of photons there from and a computerized control mechanism
further controlling same.

1

wo 02/063034 ai mumtmrnaSB^

NB, SN t TD, TG).

published:

— with international search

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AN IMPROVED SYSTEM AND METHOD FOR COLLECTING DATA FROM
INDIVIDUAL CELLS

FIELp^iiP_BA£l^QSCiII^D^FJHEJNIiF^iIlQll

cofiecting data from individual cells and, .« pflU-b. » — »*
"cousin i^^^di^ — ^«~o t

•j tu» ^wpnt invention further relates to an
Hkrrete locations in a cell earner gnd. The present invenxo

locations based upon data collected therefrom.

i - — — » u de 7 b,e

s to conduct analyses on a specific sample to compare to notmafiste V ^^^
examplcHvetenzymelevCsfiomaspocificpafienlcomparndtonom^nvo

valuesformesmeen^mom^beusedtodiognosod.abetes.
fiaauemome.m^.etmdlfionmup^ont.I.aon.^^ma,

ta ttntdyl M.cnmes such as . fluomscence .c.tvaud e=» sot.., (FACS)

be analyzed. However a FACS machine cannot

designed, inpart, to overcome*.* problem. Kow.ver.uF

teassny individual calls afier sorting. This limitation precludes both lunette

::Lo fi „divid»loe„s»dreeoveryo fi „d i vidu,ce„safier«»,b»sedupo»

K W C f o re ,anumo=ro f p,iora rt dev i c»»e re p. M .«dfiy~c,,.
maddresssom.oftheseissue, (U.S. Pat Nos. 4,729,949; 5,272,081;
'•''Tsirrt^tcucfiesmemodsandaP^msforpe^ng

„ ^ysesonindWiduallWiogce,,, Accordmg

■ a- .H„ fl l r-ells are forced into holes in a earner gnd so that each 01

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_ nroc erties of individual cells and
f nr observing or measunng one or more propera
me ans for observing means mA

control means for controlling the relative lo ^ tQ

5 mayinclndeopucalscanmngmeansfor rf ^

f tHo not disclose such a scanning means. Further, the tea g

* ^^^vahle with a scanning means.

15 patent. -j^+ification and subculture of a

U.S. Pat. Mo.5, 272,081 teaches identification an

f rp»<! residing in a grid of the type taught in U.S. Pat. No.
selectedsubgroupofcellsresidmgm gn

4729949 U.S. Pat. No. 5,506,141 is similar to U.S. Pat N ,
4,729,949. U ^ rf ^ ho , es m

except that it teaches that thepositio „ mtinthe teachings of these

^'US Pat N o.4,772,540toDeutschetal.teachesa m ethodof

I d 'rid resistant to mechanical distortion. Despite the added
manufacture for a rigid grid resistant ^ fa

™—rr~TT-

w Wrh reauires attention of an operator 01 y
results of an assay performed merely

PCT/US02/02660

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individual cells devoid of the above ttmitaPon.

automated system for loadtng .ndmou ^
lo.oWMdua.^^ona^a.^^^ *

« a .owe, surface o <h ^ ^ ^ (d) „

individta.1 cells fro™ a populate of cells P ^
^mcatton between the 1F> ^u^dreacrvoi.. Application of

vacuuntviatheportcausesthetndtvKl w
^on K n,.va to ,.^W i v i d™.diac^.oc«op*.

• w removal of the liquid from the space.

suasion inio lnd.v« *— Prions w*n -
.co.odc^pHaaa^a^otWp.aco-sO"^^

cells in suspension to an upper surface of the gnd, 1.

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. co that individual cells enter at least some of
to the portion of the cells in suspension so that indivia

^individualdiscreteloca^ present Mention there is provided an

5 belonging to a population of cells in s p fa ^ ^

carrier grid including a plurality ot indiv, ^

and retaining

„ holder W^- - * ' It*-*'"''

discrete locations, (e) a loading ^ ^ suspensl0n ,

holder containing the grid, the vacuum source, the popu!
15 and the at least one liquid reservoir; ^ ^ ^ ^

20 ton .be Individ- reading m .he .nd.v. ^ rf

fc cell « gnd *■ — -7 J the — «H*W

• ,. leas, one liquid reservoir, .he loading dev.ee ano
suspension, the at least one uq indivi dnal cells from a location

^.T*- — — " ^.cetfthecelloarHer
25 ~ - — » f ^ ^"nannnioaHon vdth the

space- a fAp^t invention there is provided an

Acoqrd^.osd.l B »«.e.»P«o.of^P-».J'

j A a nfrollection of data from a plurality ol maivi
automated method of collection ^ ^ (&)

to a population of cells in suspension. The method comp

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. c.11 earner grid including a plwali* of individnal discrete mentions .

ho ,dnr soch .hat a tower s«rf.ee of* gn fc

( ^™ S . n * wito , di5CteKtalit ,» bym.ansof,
ce„s in suspension ,o move *. I. -»* of ^ „

soun* =— Ma «o <h= po* W W« «" one
■™v t.l allowing communication between the at least one hqma m
rT^ell^^Waida^at^Utep^^otc.Ba^

'° '^TI.rr^c.m^in^WMdua.d^^oaa
suspension while the inmv t „ wi *h the individual cells from a

i ^ta the eroup consisting of the space ana an u PP
^select dfron^th^

cell earner grid; and (f) employ g ^ ^ rf

„ between the grid holder containing the grid, the

• ™a the at least one liquid reservoir; (g) illuminating in

• j u^M-r the vacuum source, ine
2 „ actions of the oe.1 enrene, gnd holder, the v, a^ptica,

^nsion, the a. least one Hqtnd msecou, the loamng

rxtrrco^ofi^ap^ofh.^^

article of manufacture uselul ior co

w;™ of cells in suspension in a clinical setting.
» ^^'^^Lrgridino^apWltyofWWd™.

^'^^^nndL^ngonaof^-aidn^^grid
locationatscapableofengagmg ( .e„ ri ji. in communication t»ith a

held in a *id ho>der snob ma. a low snrfaceo - . .
30 space within me ho,der, <b)me ce,U„ner gm, bolder,

ix\ «t least one liquid reservoir fox bringing ax
connectabletotheport;(d)atleaston q Usin suspensi on while

into contact with the individual cells from the population of

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, •„ , ce llsresideintheindividualdiscretelocations;(e)aloadingdevice
facilitating communication between g res ervoir, (f)

tan 0* Wividaa, «Us searing » <ha tctions of

,heatleastonehquid.eservo,r,.heloadmg device „|„„face
A pp,ic..io„ of vacuun, via the port causes the ndm

Mi.U^-.t,— * I Ursrtd. The grid holder
impr ov«ri e.aehc-opdca! scaler e.pah.e «J

,„. grid composes . may of d.so. — ^ rf

capable of eagaginB »d re»m,ng a s»gle hvmg oe. , W

— ~ rrri.:— —

for co-ordinating actions of the optical

the cell manipulation device. 4nventio n there is provided a

• ♦ ciniarfditional aspect of the present invention mere v

, n According to still additional a y of individual

, • ^f mm individual cells belonging to a plurality of indivi
method of collecting data from indiviau

• ^ r locations by means of an improved electro-optical
cells residing in predefined locations oy

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discmK !«-»» » f »" ^ comptte a camera, the

, i8M soorca, a ^ - " dividua , eens «i„ a ceU

md ane.com* -W--*-» — f*"?; omacon«o.o.i.,rhe
,„ and. d.ccnn^p.l.^^™ 14 " — - "<""

control nnit comprises a compoler. ^j^^h of mo invention

Acc„rdi»g.o^erfe.mres,np,.fcme em* *

described Wow, «•= Srid holder is const™*, of a. leas. 0^

„ groupconsisnngof Lucito, pWc ^^JUned embedments *.
According to still farther fenn.es mflKdeson

—*T*" ^ B , te ridho,deri„..thc
20 l^sdnvf^Wremov^thn^dhold^ dhoitotonl]M
fte grid hold., » . scanning assay devrcc, and (,v)

scanning assny device. a-rfbrf preferred embodiments the

According .0 still further feature* » «« » rf „

25 , ras ,o M ,o to rio B m,..ioas«nn,.onv.yo,be 1 «,..loa S .o

oocpistontmd a. Las. one rotating pi* ^ ^d embodiments «h.

-

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«Lrce theatleastonepopulationofcellsin

pension, the ^^^^^p^-*^*-
According to still further features - th ^ ^ ^

systemf^ercomprisesacomputenzedc- atlea ^ one Ration of cells in

According to SOU fW" 1 " lea rf fl „otescOTc« .s

^ tan »* « ° f ; » ,„„ s*s living *

u census »P— f "" ^ lov) *'

Accocding » ^ ' ^ in te individual discr...

20 Accorfrngtosulltom"'"™ 1 ^.wrt

Acceding to sdU fl"*" " * fished »i,h th. of

aep o f p 1 - i o g «..^ ta .d-n.oo,.l°o^^' S

A.»*S.o-ll^ tepsdKtedtrom ,h. 8 roup
„„U,od tad*. " toS ' °° ^ loadtais d.vi..i 00

scanning assay dev.ce, rs P ^ ^

3 0 configured for performmg M * ^ preferred pediments

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10

^^^^^^^^^

h °' d "' fi,rf*r features in the described prefemd embodiments

removdoftlteliqoidfiomthespace. embo diments the

BHer, end » . scanning »nt. capable of '*> am * ^ uni , m

^ tight sonrca. The optica. »i. and -mponenta ««of

^.p consisting of a nricr.pip.de, a needle, and an elects
. .-^ c nftV.e cell manipulation device.

Aooording to s.,11 fodher featn-s ,„ ^
Scramble of an action selected iromtnegiv v
mi cro P ipetteis capable dualcell> moving at least a portion of

leastaportionofanorganelle^an^d idud cell ^ one of the discrete
the indidual cell's cytoplasm, and removing the rndrv

locations. , nreferred embodiments the

According to still farther features in the descnbed preferred
25 According t .H from the group consisting of injecting a

substance into an individual cell residing m the discret

substanc e fr omanindi V idualcell^

— ^^rr^oupconsistingofapplyingan
30 electrode is capable of an action selected from ^ ^ P &

potential difference across a membrane of an indivi

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location, and creating a potential difference across a membrane of an individual cell
residing in the discrete location.

According to still further features in the described preferred embodiments the
electro-optical scanner capable of collecting at least a portion of photons emanating
5 from the individual cells residing in the individual discrete locations is further capable
of gathering polarization data pertaining to the photons.

According to still further features in the described preferred embodiments the
polarization data is useful in making a medical diagnosis.

According to still further features in the described preferred embodiments the
10 method comprises the additional step of providing at least one robotic mechanism.

According to still further features in the described preferred embodiments the
at least one robotic mechanism performs at least one function selected from the group
consisting of: (i) placing the grid holder into the loading device; (ii) removing the
grid holder from the loading device (iii) transferring the grid holder to a scanning
15 assay device; and (iv) removing the grid holder from the scanning assay device.

According to still further features in the described preferred embodiments the
method comprises the additional step of including within the electro-optical scanner a
cell manipulation device selected from the group consisting of a micropipette, a
needle, and an electrode and the control unit further co-ordinates actions of the cell
20 manipulation device.

According to still further features in the described preferred embodiments
the step of illuminating the individual cells residing in the individual discrete
locations and collecting at least a portion of photons emanating from the
individual cells residing in the individual discrete locations further includes
25 gathering polarization data pertaining to the photons.

According to still further features in the described preferred embodiments
the article of manufacture further comprises instructions for performing specific
analyses therewith, the instructions reducing the need for calibration thereof.

According to still further features in the described preferred embodiments the
30 article of manufacture further comprises a cell manipulation device.

The present invention successfully addresses the shortcomings of the
presently known configurations by providing an improved system and method for

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conacling da,, from individual eel,. .„d, mote partly, » a— . of ft.
p^a. of causing 0.1,3 to teaid. inindividna, disc™., locations and of addmon of
b. -on**, process lo of dala from ool.s aiding in MM

aiaoate .odious in a 0.1. crter grid. The pteaen. invanlion further rotates lo an
5 arti.1. of m.»uf.c.u« which indndos sn el.«ro-o P „o.l « -11 cnm« gnds
md a lo.ding device fo, — . The pr.s.n, inven.ion forth* rel.tes «o systems
and m.lhod. which allow recover of specific cells aiding in individu.1 d,sc« e
,„c.,io„ S b»«d upon date collected Ihenuron, Th. pros*,, invenlion suceesafiuly
address forth* shortcomings of previously known eonfigutelions by
inoon.or.nng - oplid short* inlo m etecteo-opfio.1 seiner in orde, lo ptev.nl
bleaching and biological damage.

gpjpcTVF SCR TPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with
15 referencetotheaccompanyingdrawing, With specific reference now to the
drawings in detail, it is stressed that the particulars shown are by way of
example and for purposes of illustrative discussion of the preferred
embodiments of the present invention only, and are presented in the cause of
providing what is believed to be the most useful and readily understood
M description ofthe principles and conceptual aspects of the invention. Indus

regard no attempt is made to show structural details of the invention in more
detail man is necessary for a fundamental understanding of the invention the
description taken with the drawings making apparent to those skilled in the art
how the several forms of the invention may be embodied in pracuce.

-,5 In the drawings:

PIG 1 is a schematic representation of essential components of one
embodiment of an improved electro-optical scanner according to some aspects of

the present invention;

FIG. 2 is a diagrammatic representation of a physical arrangement of parts
30 in an improved electro-optical scanner as shown in figure 1;

FIG. 3 is a;

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10

FIG 4 is a cross sectional view of an automated system for loading
individual cells from a population of cells in suspensi and for transferring the grid
holder to an assay device according to the present invention;

FIG. 5 is a flow diagram of method steps according to the present

invention; m

FIGs. 6a-i illustrate different embodiments of cell manipulation dev,ces

according to the present invention ;

FIGs. 7a-e illustrate different embodiments of robotic mechanisms

according to the present invention.

ppcr pTPTiON OF THE PRE FERRED EMBODIMENTS

The present invention is of an improved system and method for collecting
data from individual cells In particular, the present invention relates to
automation of the process of causing cells to reside in individual discrete locations
and of addition of that automated process to collection of data from cells residmg
in individual discrete locations in a cell carrier grid. The present invention further
relates to an article of manufacture which includes an electro-optical scanner, cell
carrier grids and a loading device for same. The present invention further relates
to systems and methods which allow recovery of specific cells residmg m
0 individual discrete locations based upon data collected therefrom.

The principles and operation of systems, method and articles of
manufacture according to the present invention may be better understood with
reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, rt is
, 5 tobeU nderstoodmatmeinventionisnotlimitedinitsapplicationto

eonstruction and the arrangement of the components set forth in the following
description or illustrated in the drawings. The invention is capable of other
embodiments or of being practiced or carried out in various ways. Also, it is to be
understood that the phraseology and terminology employed herein is for the
30 purposeofdescriptionandshouldnotberegardedaslimiting. Specifically,

embodiments of the present invention will often include commercially available
components. One ordinarily skilled in the art will be capable of selecting and

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^mblingsechcomponen.s. Daails of specific comply available
ofanal agooscompono» K o m oas i ,ybe.ff.o t odwiO M ». S ob M «.llyal,«nn 8 fi«

inVe °" Referring now to <be drawings, figure 4 illusfia.es an automatod ays..™ 70 for
dW looa.ions 7, wimin . an,y ofiodividual discre.e ,o«,,ons 77 tooaftd ,

s „obme«a.ower S « 6 c«80ofgrid5i» in common, cafion w.ft . space ,2 w*n
bolder 71. Sys.em 7. ft*, in.md.s a — source 74 conneclable to a por. 73b.
S yst em 7, Id. includes « W one liquid reserve,, 75 fo, bringing a. leas, one
liv ,ia ta tooon.«.wi.b te indiv i d«alee,lslro J »,popu.a 1 ionofc«,l S 8, m _

S^^fn^inoK^a^ingdevice^faeUi^e™— «—
pid bolder 7, confining grid S, Venn™ so»c. 74, popnlafion of cells 81

m a ,3b „ picmreO) causes ft. individnrd ee.ls from .be populabon of ceUs 8 ^
, s u s pensionto mOT e irt oftei„d i vid»ld i s n eto,oc.bo M 77. ^onebqum
w be applied to *e individual cells from a locafion se.eo.ed fion, ft. group

■ „ co^nnnicfion w„b lower surfcoe 80 of grid 5, a. .east one po„ 3 («wo ports 73.
o^epictored, formftoducU.nofalMd into space ,7 and for removal of .be

25 liquid from space 72.

Acconb„gtoprefe m de m b„d ta en.offteinve„Uo„po«73,»olutofi n .

p „ rt 73a S erv i u g f,,in«oducfionofali q ,ldintosp,ce7 2 »ds=condp<.r.73bs^n,g
L removal of ,be liquid fion, space 72. Liquid reservoir 75 and vacuum source
„ be connected to pons 73. and 73b by conneering means 79 a and b
30 respeenve,,. Conneering meons 7, may be, for ™ple a mbe, a pipe, asleev...
gasket or a flange.

0

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System 70 may further include at least one robotic mechanism 78.
tova rtoIemb.dlme„«so t ,ha i uveu,lon, ro bou.mecha„,sm78,sdes, B „«a»d

include, but is not limiled », d« "ho™.

Placing 86 (figure 5) grid holder 71 into load.ng d.v.ce 76,
Removing 91 grid holder 71 from loading device 76;

Wening 92 grtd holder 7, to a — g assay device 25, and
Removing 101 grid holder 7, from the scanning assry dev.ee 25^

• ^ a* f^rPxamDle at least one robotic arm 60 (figure
Robotic mechanism 78 may mclude, for example,

. ff - ltmre 7b) at least one pneumatic tube 62 (tigure
0 7 a),atleastoneconveyorbelt61(figure7b),a P

7c- arrow indicates direction of flow), at least one p.ston 63(figure 7d),
additional embodimenta of the mventron, compntenaed

on e li^nedLrein which ia oapable of imparting • — "
20 fl „„„ s oeucet„cell ! 81...tleaato»ev»velengd..

TheaUeaafonereagentcnpableofimprntingameaaurahledegmeof

™vbe fbrerrmpleasuoatmatethmmfferentiallysttmsl.vmgcells.
flnoresoeuce may be, for example measurable

■ cells Alternately oraddiboMlly Ore a. leaa. one reagent capable of

nucleic acias. «. fi olir escently labeled antibody.

.^oree of fluorescence may be a flourescenuy
" l—n is further embodied by - au.omafed memod SS (figme

„ sUorloadingindividurdoellsfrompopuladonofoelMlinauapensionrnto

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10

, t A - m cell earner grid 5 contained in cell carrier grid bolder 71. Method 85
82 of arid 5 and automatically applying 89 a force to

.nechanisn, 15. According* soma pref.-a embodnnents, *= -=P ™

engaging and <- of * "Mdual — ■ « 5 " Wd "* """1 71

* 1 « M. — holda, 7, and v.cuun, — T4 — * « ^ »

includes a loading device 76 facilitating communication between gnd holder

least one n 4 u. suspension to move

73 causes the individual cells from the population of cells 81 m P
25 rLdu^iscretelocations.. S^eS^erinc^an^
scanner 25 capable of illuminating cells 81 residing in locations 77 and
least a portion of photons emanating therefrom.

Figures 1 , 2 and 3 illustrate an improved electro-optical scanner 50 a cording
„„ Scanner 50 is capable of individually collecting data from a
to the present invention. Scanner P , n(j77 Scanner 50 includes

optical unit 6. Components of optical unit 6 include, but are

30

an

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t0 - aCameia( * n.^.n an optical ah»»er(Q switch 24; figure 2),

4 integrated photomul>.pltere(PMra) 11), an oprrc . , , ...

llL«on e op.«fi..er22(fi 6 ure2, Scanner SO may filrther include a cell

t Jo tl mprev.r„c„«iu.he»«.fo,us.»iO.»can„«50. Scanner 50 further

■ ™i, a 2 .»d 31 capable of uxposins discrete locaiona 77.0
includes a scanning unit (1, z, ana

,0 light fromlighl source 14. . „„, The

The XV .able driver c«d 17 of conholler 15 eontrola the XY surge 3. Tn
Z 2 stag. (Newport stage M-426, Low profile croaaed roller berning tranalahon
l g .LcMA-.2PP12.5nu„ B avelopenloops,«pp=rCMA.c».o,,,s

„ USA tot.dintheelectroniobortl!.). The coarse Y stage 1 (MICOS, MT 65
l^insu.l.ainisc.o.olser.movere.ntofall.hes.ege, Specrfic

• tinn but rather to aid one skilled in the art in practice thereoi.
20 mV "TlldUSin^^of.c^laprevidedfiir— fing

eonf.gure.ion of Oris conrpute, include an easy » use grephroe. nae, tn«*e
romne W or k i„gcap.b,liUeaf«,d..,a,oregea»dWera„dc.pac, C ,,o

^ldi^.^onsc.uifiW^p.es^ingina.^0-^
employing scanning unit (1, A ana ^ °

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count, two tteprua.e w.vel.ngUvt) opted ahute 24, - „ let*, one ^ « 22.
Ligh, source 14 may be switched off, turned en, and connote, « reapec to
htertrH, by 10 contrete 18. Cooretnaung of acrions of .ho optica, urn. «, the eel,
earner 5 and the scanning met (1, 2 and 3) is b, a control unit 15 indudtng «

,„co O t,olsc» K .50.Th=fh3.c»dn,. y bc,fo,exa mP le,a P CL- 8 3616
second card may be, for exantp.e, » XV stage drive, card 17 (ISA bus, d,g.W

Wldhronn, Germany). The third card may he, for — * an K> — 18 that
cootro.s »H datt. acquisition and con*., (Media Technologies, Yehnd, Israel).

Cel. Came. 5 is placed on the XY 3, Z 2, and com. Y 1 stages, ate .. has
h^lo^^^aa^h^-Meclr-cnlc^^l^

centers grid 5 with respec, to crane™ 9 which may be, for atcamp.o a B/W 1/3 CCD
Ca m .r.(Sony, J .pa»,or.co,orCCDoranyoUa=rdevice»hichan,blesv, CT ,„g

grid 5 for the purpose of orientation.

„ Viewing •. call loot-Ions 77 on ft. video screen .0 defines for compute 15

the location of each individual h*. Screen .0 ma, also be used in cel. retnevtd

monitoring. The opora.or riot* a field of inures, and scans the grid u s ,„g
Lro„ i c!nyco„non«ds a go S 2. n d3. F or=.chspec i fio,oc..i.n77p,mm a e, s ar.

measured. ~^~^"^£Z£L —
2 5 mea S »r e me„.,iottnsityd.t«(aam ra au re dbyPMT 8 U),»ndcelllo

patent are srored in a data file on connote 15. According » preferred

course of the scan. All stored data can be read and analyzed by numerous algorithms
presen, on comp»,e„5, or on.remoto computer. Uaer.4tnay ha, reretrmnple.a
3„ Ld-s.atediodeputnp.dl.ser LCS-DTT-362 (User Compaq Moscow, Ruaare,
wavelength -473nm; KlOmW) or a He-Cd (Licomx Inc S-n-Cre,

PCT/US02/02660

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18

CaUfomin, «SA ; -»B. accords «o — ^uirenrent, In »™

cases «. SO will fa** tors .4 whtch mtty be spccAd by a use,

to box 19 «A supply ... <be power to «he system. Alternately o,

aaditiona.ly.some eonrpon^^b. powered by oirecreo—ro.—aM

electric outlet. ^ •

The laser enters the optical system by means of a fiber optie eable 12 (e.g Oz

(Centronie^NewAddington, England, OSI 5-V-10M/10K). Detector 8 mon ors the

by the ND motor 23 (Maxon DC motor, Sachseln, Switzerland).

According to preferred embodiments of the invention, scanner 50 further
15 inclu desacellmanipulationdevice7. Cell manipulation device 7 may be, for
e X ampleamicropipette37,needle38,orelectrode39ca P ableofperformm g
actions describedhereinbelow.Accordingtolheseembodm.ents.control^lS

further co-ordinates actions of cell manipulation device 7.

, • a ■ - n i n fho form of cell retrieval unit 7 (Eppendort,
Cell manipulation device 7, in the torm oi ecu

, *v»<* yy ^tflpe 3 After a specific cell location is

20 Cologne, Germany)islocatedneartheXY stage3. Atter p

• sc JrSCoraltematelyandpreferably, by controller 15 based upon an automated

analysis of data. ^ .

offluorescent-polarizationinhvingcells. Prior to measurement the system
establishes the grid orientation as detailed hereinabove. Ihis establishes an address
for each discrete location in cell carrier 5.

Use, radiation wavelengths of, for example. 473tn», 442n» o, 48 8 m» «
M aelectablebyanop^orofsca^O. A po to 3Z divides the

0) e m »a 1 .n gfom d.ecell..o t wob«».a(I, m d, I ,.vrhicbtne i b n h«,po.ar,a«d.o

PCT/US02/02660

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19

two separate orthogonal planes (parallel and perpendicular). Each of the two
polarized signals is later divided into two (WL, and WL, where WL indicates
wave^FourPMTsndetectand read eachofthe four signals at photon —

mode.

Polarization and fluorescence intensity are then calculated by a computer

according to the formulae:

Degree of polarization

Fluorescence intensity

taft ^ y ,, te s yS «™«ec l sch» 8 =so ffl »o^«ncepo lmM ondepo^o„)
and floored i«* depending on bioohemioal oondUions of .he c,U.

The op.ieal sya.em includes » excfl.fion subsystem 100, a fluoresce

aobsys.em .proton Astern ,20 - . — • » * *-

calibration.

Excitation astern 100 W»k. laser light source 14, as dettrlcd

System 100 further deludes an op« *— I*— » <™ .

filter 26 (Linos, Goertingen, Gennany), part • 371142, r -10%.

Olher components of subsystem 100 are Lens 28 (Linos, Goeningen,
2S Ge m a»ypan#3n247,,dichroic mto ,r 3 0«.45-»ng.e(O m e ga opfic al ,Br,n,ebo ro ,

USA part # XF2010 (505DRLP), reflection spoofial „„ge<500nm ; ,,— »

( Or,«,,S.rt,ford, U S A part#2 6 3 5 0, ,«d

Lhnrgm 33 0.8mm, Pl.r. bean, aplitter 34 (R.ynard Corp., San Clemente, USA
-dBHrtSH, r-85-/.;H«ree reprints emission and r represents
30 „fioofio„),Pho.od«eo,o,3<,(Con t ,o„ic, N e»Addi„ P ,», England), and mientscope
objective 40 LWD CD Plan 40* dry (Olympus, Hamburg, Gemrany ).

ND filter 22 and photo detector 36 ant responsible fo, keeping the laser
inle usi<y » . con**, level. Q-™«ab 24 mgoltdes Ihe dnm.ion of laser exemafion

PCT/US02/02660

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exposure, This prevents bleaching and biological damage and allows measurement of
cells which might exceed the maximum measurable excitation in systems whu* lack
an optica, shutter. This is achievable by setting a maximum number of photons and
.ecordingatimeatwhichthismaximumisreached. Cells which reach this pre-set
m aximum can then be ranked according to time, those having the shortest tame
exhibiting the strongest excitation..

ND filter 26 provides additional lowering of laser intensity. Lens 28 and
m icroscope objective lens provide a laser spot of 1 8-20 microns at the cell earner 5
plane (figure 1). Dichroic mirror 30 enables passing of laser excitation in one ^
direction and fluorescence emitted from the cells in the other direction. Polanzer 32
pmvidesahigher extinction ratio of polarization. The beam splitter 34 drvrdes the
iaserexcitationbeam into two. Most of the energy enters the microscope objective

and regulation-

Fluorescence subsystem 110 includes microscope objective 40 winch may be,
for example LWD CD Plan 40X dry (Olympus, Hamburg, Germany). Objectwe 40
collects fluorescence radiation from the cell. Further included in subsystem 110 ,s
plate beam splitter 34 (Reynard Corp., San Clemente, USA , part# 880.; 1, *-15 A
,=85% Beam splitter 34 directs this radiation towards field diaphragm 33. Freld

0 diaphragm330.8mmres*^^

rlasTglecellatatime. Cube polarizer 32 Oriel, Stratford, USA, part • 26350)
divides the fluorescence beam into two beams that are polarized at two orthogonal

PlanCS ' Further included in subsystem 110 is 45' dichroic mirror 30 (Omega optical,
25 Brattleboro, USA, part* XF201 0 (505DRLP)) with a reflection spectral range <500nm
and a transmission spectral range >500nm. The two dichroic mirrors 30,31 prevent
unwanted laser reflections.

System 110 further includes flat polarizer 42 (Melles Griot, Irvme, Cahfonua,
USA part # 03 FPG 001) to increase the extinction ratio of the two polarized beams.

further divide each of the two polarized beams into two. Two flat mirrors 46a,b

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2)

10

20

(Linos, Goettingen, Germany part # 340083) aid beam splitters 44a,b in directing the
four polarized fluorescent beams toward the emission filters 48a,b and 51a,b.

Subsystem 110 further includes emission filters 48a,b and 51a,b. These may
be, for example two pairs of Omega optical (Brattleboro, USA) filters part # XF3022
(580DF30) and XF3007 (535DF35) or two pairs of CVI laser corporation filters (part
# F10-510.0-4-l.00 andF10-510.0-4-1.00; Orlando, Florida, U.S.A.) Choice of
filters 48a,b and 51a,b will depend upon the specific embodiment of scanner 50.
Emission filters 48a,b and 51a,b transmit fluorescent radiation of the required
wavelengths toward each of the four PMTs 11.

Projection system 120 includes illuminating halogen bulb 4 (figure l)(Heine
XHL, Herrsching, Germany), part # X-02.88.044. Bulb 4 illuminates cell carrier 5
(figure 1) on movable XYZ stage 3,2.

Projection system 120 further includes microscope objective 40 LWD CD Plan
40X dry (Olympus, Hamburg, Germany). Objective 40 produces an image of cell
carrier 5 at the reticle 52 (optical target in the objective image plane) plane
(magnification up to 40X).

Projection system 120 further includes plate beam splitter 35 (Reynard Corp., San
Clemente, USA ), part # 845.1 , x =85%, r=15%. (The symbol x indicates transmission
and r indicates reflection)Plate beam splitter 35 and IR LED 53 are used for
illumination of reticle 52 at the orientation of the cell carrier 5 (figure 1). IR LED 53
may be, for example, an LED supplied by OSRAM Opto semiconductors ( Munich,
Germany). Projection system 120 further includes reticle 52 (Linos, Goettingen,
Germany), part # 391 130. which is the optical target for grid orientation.

Projection system 120 further includes lens 54 (Linos, Goettingen, Germany),
15 part # 3 1 1338. Lens 54 projects images of the cell carrier 5 and reticle 52 image to
CCD camera 9.

Adjusting eyepiece 130 includes flat mirrors 46c (Linos, Goettingen,
Germany), part # 340083, lens 56 (Linos, Goettingen, Germany), part # 311310 and
lens 58 (Optosigma), part # 01 5-0040. Flat mirror 46c directs the light beam from the
30 field diaphragm that is illuminated by the halogen bulb toward the lenses 56 and 58.

PCT/US02/02660

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22

Lenses 56 and 58 together with user's eye produce the field diaphragm image.
Eyepiece magnification is preferably approximately 10X.

During use of scanner 50 a laser beam passes through ND filters 22 and 26
and lens 28. The beam is subsequently reflected from dichroic mirror 30 and enters
polarizer 32. The linearly polarized excitation beam, passes through the field
diaphragm 33, reflected from the beam splitter 34, passes through the microscope
objective 40 and illuminates one cell (that is in the center of the field of view at the

time of measurement).

As tire cells fluoresce, they emanate photons which are collected by the
objective 40, reflected from beam splitter 34 and passed through field diaphragm 33,
which restricts the field so that only one cell is read. The photons then reach the
polarizer 32. Here the fluorescent beam is divided into two separate beams that are
polarized in two orthogonal planes.

The first polarized fluorescent beam passes through two dichroxc rumors 30
, and3 landisfurtherdividedintotwo,bybeam S pHtter44b. This pair of beams
re aches the emission filters 48b and 51b while only one of the beams is reflected
from the flat mirror 46b. .

The second polarized fluorescent beam that is reflected from cube polanzer
32, passes through flat polarizer 42, and is divided into another pair of beams by a
0 second beam splitter 44a. This pair of beams also reaches the emission filters 48a

is reflected from flat mirror 46a.

Orientation of the cell carrier 5 is achieved by the projection system.
An image of the cell carrier 5, is projected fiom the objective's imaging plane
25 (reticle's 52 plane) onto the CCD camera, by the lens 54. Magnificats m=-l . (The
mi nus sign means inverted image magnification). Simultaneously the same lens
projects the image of the reticle 52, which is illuminated by IR LED 53, onto CCD
camera 9. (Magnification m=-l*)- This causes two images to appear on vuleo
m0 nitor 10. The first image is a movable image of the cell carrier 5 and the second
30 image is an unmovable image of the reticle as a background.

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23

System 65 forth* comprises a oompu.erized control mechanism 15 destgned
„d configured to co-ordinate actions of grid ho.dc, 71 confining grid 5, vacuum
^ popnlationofcallaSXinauapenaion, li q uid reservoir 75, .oading devtce 7C
ma e,.ctro-op,io,l soaane, 50. Th, a, M . liquid ma y no
MMM oolls aimer from space 72 o, from upper anrf.ce 82 of gnd 5. Gnd holder

oflimnd from space. (Port 73 is pictured as two porta 73, and 73 b.

The presen. invention is further embodied by an automated memod 84 of
Section of d» nom a plurality of individual cella belonging ,o a population of cells
„ glinsnspension. Thememod includcsme steps of providing 83 eel, o m =rgrtd 5

each ofindividnllocarions 77 iao.p.b,eof engaging -ndretammg one cells 81, and
h oldi»gu»gridinagridh»lde,77»chma„ow.rsn I i»o«8.ofgrid5 1 am

I mcalw i ,hap.o.72wimmho,der, 1 . Mem„d84 ™*>^°»^
„ lowing 87 atleaa, one ,i,uid to en.e, and leave space 72 in holder 71 v„port73
ll^of caualn S^-idna.eenafbampopmadon of o.^o mo.

i„,„ the M. 77 by me«.a ofvacuum samroc 74 conneetable ,. port 73. . Medmd
84f ^, i nc,udeames 1 epof»pp,yingmepopn,a,ionofce,,s8 1 maoape.mn»nd

Tbeat.e.s.onebr.mdmayoommnnicatevdmmeindividua^Uae.Oter nomspace
,2 or from upper aurfaoe 82 of grid 5. Medmd84 mrthe, ino.udea the a.ep of
employing , loading device 7a to MM. commutation bortvean gnd holder 71
conlming grid 5, v«uu„ source 74, population of c.,U 81, and l,<,utd reservotr 75.
25 M«hod L «=■ includes me atep of Illuminating ,5 me mdividua, oe„s restdmg ,n
inaividu, disemte .oeariona 77 and collecting a. leas, a portion of photons „

sK pofoo-ordi«aUng.c,ionsofgridbolder7.,v— source 74, pop^on of
ft 1 iq nid reservoir 75, loading device 76 and electro-optic,, scaoner 50 by mean, of
30 a computerized control mechanism 15.

PCT/US02/02660

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24

use*, for collection of da,, ftore , ,M of individual -» bolongtng . .
useful tor con „„MinicM. setting. The article of manufacture

,4, »*. reservoir 75 , loading device 70 elee^optica, scare,., 50 retd
eorep».en»deo„,re.reeoh m isre 1 5, S de S ertb=dh,rei,»bove. Prefer*

n 81 to move into individual discrete locations 77.

(Pi^s ,, 2 and 3, capab,. of individual,, collecting data ftore ap«»» ^

optical unit The optical unit includes camera 9, light source i«, p
optical unit. d 26 Scanner 50 scans a cell

I5 optical shutter 24, and at least one optical filter 22 and ner50further
• -a * as describedhereinabove. In order to scan grid 5 scanner 50 farther
camergnd Sasdescnb • gthediscrete locations of grid 5 to light

includes a scanning unit capable of exposing me

Chgh.soure.a4. Scanner 50 rerth.r includes a cell reanipul.Uon , ev.ee I «
, . „.V> Scanner 50 further includes control unit 15 whteh tncludes a

erebcdireenl.rnethod 04 of ao„.o.ing data iron, individual eel. belongtng o a

Laleotro-optieal scanner 50. Method 04 Modes the steps of caustng
unproved eleotroopuca. „. a, ,„ t,. „j,a R ed and retained in

individotdoellsfrorettteplurelireofindrndualcellsSlto beengag

rapplyingforeereeretoOO. Method 04 further inc.udesree sf.p of exposreg*=
3 „ ^ToLonafohgh.^^.lighfaoure.UWerep^ngn — gassa,
SOandtheatcpof gonere.ing data ftotn an optical unit na deaenhed

II

PCT/US02/02660

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25

m*+m. Memod 84 M. inches ft. step of * — —

from the plural*, of MM*- coll- »» «» • «" -""to*"™ ' " .
teaoflbedneminabove. M.,h.d84f»rth.,inc ! , rf . S fl«,,epof ^rdinatog a.uona

iooividua, disc„.« ,ooa,,o„s Wo conKc. 90 with a. leas. - dehve^d, to
^..f^^d-rvcirTS. M.d,od84„,.y include

bolde, 71 ton, sc«ning -ay device 50. These steps may be performed,
example by robo.ie meehanisn, 78 designed and oonfigrned to that pmpose.

■ ,, j-n;,,,, 87 ,oace 72 and automatically applymg
Preferably, the steps of automatically filling 87 space 7A an
a force 89 » accomplished by causing a liquid .0 flow 93 through port 73 in gnd

" ^"Lrdingroprefenedemhodimenls of fire invendon, ele.to-op.ic,, scanner
5 . i „. 1 „de S anopfi.a.um.wy.h,„..nd.s^.m9,,igh.sou«. 1 4pho,om*p 1 . M

„, opto*, shun., 74, and opdcd fiher 22 Elecfro-op.iea, scanner 0

fr^Hno.udea.sc.nninguni..,^ eapahieof expoaing diacre.e logons 7,

20 » Ugh. torn .igh.so.nc. .4. The op.ioa.uni, and component .hereof an ,h.
s ealg«ni.a re .ontol 1 eflh,compn.eH K d.o„to,n,eeh«nsm,5aade« 1 d

teinab ov, meeto-opflcalaeannerfiO may tome, include , eel, mnmpulafion
39. ,„ and. a ease, .onto, uni. 15 forth., co-ordinate, ae.iona of ce„ mampnlaton

" ^MienapipeUe 37 may he empioyed, to example ,0 remove 97 a, leas, a portion
of an orgimene torn . individoa, cefl. Figure 6h i.luato.es remova, of cefl nucleus
45 torn . cell afle. micmpipede 37 has pene.m.ed cefl membrane 41. Aouordmg to
M embodiments of me invention, o„„ genomic DNA is removed torn nr*,e»s
,„ 45. Al, OT a.elyo,addi.ionalMfigure6=),a.l-..portionofu«i»d,v.d»a,.eUs

PCT/US02/02660

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26

• «h**t Alternately or additionally (figure 6a)
cytoplasm 43 is removed by micropipette 37. Alternately

• tte 37 removes the individual cell from one of discrete locates 77.
nucropipette 37 removes & ^ ^

M«»dle 38 may be employed for injecting kpz™*

Needled may v extrac ting (figure 6f) a substance

an individual cell residing in discrete location 77 or extracting (
5 from anindividualcellresidingindiscretelocation77 ^
Electrode 39 may be employed for, for example, applying an el

• i* :„^rrete location 77, measuring (figure on)

==-========== -

15 isnsefulinmakingamedicaldiagnosis. rf
Methods according to the present invention may include tn
Methods ac mechanism 78 may perform

• „ oi orid holder 71 from loading device 76, transternng ? &■

• — — — rtr^^ri

calibration thereof. Alternately or add»tionally,tn

cailD , . . . „, a , described hereinabove. .

fi^er include a cell manipulation device 7 as descno

further incmoe described in conjunction with specific

Although the invention has been described in
k , entstLof itisevidentthatmanyaltematives,modificat,onsand
30 embodiments thereof, AcC ordingly, it is intended

variations will be apparent to those skilled ro

PCT/US02/02660

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27

* cnorace oil such ai.cma.ivcs, «M-ta. -* — *- « ™' hi "
spirit and toad scope of the apposed claims.

Ml publtanions, patent and pa.en. appto.ions monnonod m b.
s^if.canon^hcnsininco^ra^inkircnri^bys.fenncain.otl.a

by seferenc. In addition, citation or idon.iSca.ion of m, «fc«no« ,n dns
liic.tions-inot^oonsnn.dasast — o.atsncri^ce.savsdooic

as pribr art to the present invention.

PCT/US02/02660

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28

WHAT IS CLAIMED IS:

1 An automated system for loading individual cells from a population of
cells in suspension into individual discrete locations within an array of individual
discrete locations located in a cell carrier grid contained in a cell carrier grid holder,

the system comprising:

(a) the cell carrier grid, the grid held in the cell carrier grid holder such
that a lower surface of the grid is in communication with a space within the holder;

(b) the cell carrier grid holder, the holder comprising:

(i) said space in communication with said lower surface of the

grid;

(ii) at least one port for introduction of a liquid into said space;

(iii) said at least one port further serving for removal of said liquid
from said space;

(c) a vacuum source connectable to said port;

(d) at least one liquid reservoir for bringing at least one liquid into contact
with the individual cells from a population of cells in suspension while the individual
cells reside in the individual discrete locations; and

(e) a loading device facilitating communication between the gnd holder
containing the grid, said vacuum source, the population of cells in suspension, and

said at least one liquid reservoir;

wherein application of vacuum via said port causes the individual cells from
the population of cells in suspension to move into the individual discrete locates;

wherein said at least one liquid may be applied to the individual cells from a
location selected from the group consisting of said space and an upper surface of the
cell carrier grid.

2 The system of claim 1, wherein said grid holder is constructed of at
least one material selected from the group consisting of Lucite, plastic, and glass,
silicon metal.

t)

PCTAJS02/02660

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29

3. The system of claim 1, further comprising at least one robot*
mechanism.

4 Tie system of claim 3, wherein said a. leas, one robotic meclmism is

consisting of: .

(i) placing the grid holder into said loadmg dev 1C e;

(ii) removing the grid holder from said loading device

(i ii) transferring the grid holder to a scanning assay dev.ce;

(i v) removing the grid holder from said scanning assay device.

5 The system of claim 3, herein said robotic mechanism includes at
rotating plate.

6 The system of claim 1, »»e«i» said port comprises . firs, pod serving
for Marion of, in,. »d space and . second port serving fo, removal of
said liquid from said space.

, The system of claim 1, farther comprising a computerized control

one liquid reservoir.

. The system of olnim3, farther comprising a computerized control
^a.^on.p.pnl.tionofcaUsinsnspenrion.saidloadinsdev.caarde.laas,
one liquid resovoir, and said at least one robodc mechamsm.

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9 The system of claim 1, wherein at least one reagent contained withm
the cells in the suspension at at least one wavelength.

10 The method of claim 9, wherein said at least one reagent capable of
impart ingameasura b le degree of fluorescence is selected from the group cons.stmg

° f ' (al a substance that differentially stains living cells;

0,) aprecursorofafluo^ ^
cells;

(c) a fluorophore that stains nucleic acids; and

(d) a flourescently labeled antibody.

, , a,, mMmM n,«hod fo, loading individu^ cells from a pop»tetk» of
ce lls „ sospension 1*0 individual disc,*, h — - » - -* ?

the method comprising the steps of:

fa") placing the grid holder into a loading device;
S Lomaticanyf.llingaspaceinthecenca.ier

such that said liquid fills the individual discrete locations;

(c) almaticahy adding a portion of the cells ,n suspens.cn to an upper

SUrfaC 7r— yapplyingaforcetosaidportionofthecellsinsuspension
so that individual cells enterat least someofthe individual discrete locations.

12 The method of claim 11 .further comprising the step of:
(e) hringing the cells in the individual discrete locations into contact wrth
at least one liquid.

13 . The method of claim 1 1 . wherein said step of placing the grid holder
into a loading device is further automated.

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14 The method of claim 11, wherein said grid holder is constructed of at
least one material selected from the group consisting of Lucite, plastic, glass, silicon
and metal.

15 The method of claim 11, wherein said step of placing the grid holder
into said loading device is accomplished with the aid of at least one robotic
mechanism.

16. The method of claim 11, wherein at least one additional step selected

from the group consisting of:

(i) removing the grid holder from said loading device;

(ii) transferring the grid holder to a scanning assay device; and
(in) removing the grid holder from said scanning assay device;

is performed by said at least one robotic mechanism which is further
designed and configured for performing said at least one
additional step.

17 The method of claim 15, wherein said robotic mechanism includes at
,east one item selected from the group consisting of at least one robotic arm, at least
oneconveyorbelt,atleastonepneumatictube,atleastonepistonand atleastone

rotating plate.

18 The method of claim 16, wherein said robotic mechanism includes at
least one item selected from the group consisting of at least one robotic arm, at least
one conveyor belt, at least one pneumatic tube, at least one piston and at least one
rotating plate.

19 The method of claim 11, wherein said steps of automatically filling a
space, and automatically applying a force are accomplished by causing a liquid to
flow through at least one port in said grid holder.

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20. The method of claim 19, wherein causing said liquid to flow includes

causing said liquid to flow through:

(i) a first port serving for introduction of said liquid into said

space; and

( ii) a second port serving for removal of said liquid from said

space.

, method of claim 11, wherein said steps of automatically filling a

space

21. Thei

; , automatically adding a portion of the cells, and automatically applying a force
are co-ordinated by a computerized control mechanism.

22 The method of claim 17, wherein said steps of automatically filling a
space automatically adding a portion of the cells, and automatically applying a force
1 co-ordinated by a computerized control mechanism which further controls said at

are

least one robotic mechanism

23. The

method of claim 16, wherein said steps of automatically filling a
space, automatically adding a portion of the cells, and automatically applying a force
« co-ordinated by a computerized control mechanism which further controls said at
least one robotic mechanism.

24 Themethodof claim 11, wherein at least one reagent contained
within said liquid is capable of imparting a measurable degree of fluorescence to the
cells in the suspension at at least one wavelength.

25 The method of claim 24, wherein said at least one reagent capable of
imparting a measurable degree of fluorescence is selected from the group consisting
of:

(a) a substance that differentially stains living cells;

(b) a precursor of a fluorescent substance that differentially stains living

cells;

(c) a fluorophore that stains nucleic acids; and

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33

(d) a flourescently labeled antibody.

26. An automated system useful for collection of data from a plurality of
individual cells belonging to a population of cells in suspension, the system
comprising:

(a) a cell carrier grid including aplurality of individual discrete locations
arranged in an array such that each of said individual discrete locations is capable of
engaging and retaining one of the individual cells, said grid held in a grid holder such
that a lower surface of the grid is in communication with a space within said holder;

(b) said cell carrier grid holder comprising:

(i) said space in communication with said lower surface of the

grid;

(ii) at least one port for introduction of a liquid into said space;

(iii) said at least one port further serving for removal of said liquid
from said space;

(c) a vacuum source connectable to said port;

(d) at least one liquid reservoir for bringing at least one liquid into contact
with the individual cells from the population of cells in suspension while the
individual cells reside in the individual discrete locations; and

(e) a loading device facilitating communication between said grid holder
containing said grid, said vacuum source, the population of cells in suspension, and
said at least one liquid reservoir;

wherein application of vacuum via said port causes the individual cells from the
population of cells in suspension to move into the individual discrete locations; and
wherein said at least one liquid may be applied to the individual cells from a

location selected from the group consisting of said space and an upper surface of the

cell carrier grid;

(f) an electro-optical scanner capable of illuminating the individual cells
residing in said individual discrete locations and collecting at least a portion of
photons emanating from the individual cells residing in said individual discrete
locations; and

PCT/US02/02660

WO 02/063034

00 a compmerized «-»> md 0<>nfi6 " red * """"l"""

--■""■'"-■'Trr

electro-optical scanner.

27 The system of claim 26, wherein said electro-optieal scanner comprises:
an optical unit, said optical unit comprising a camera, a light source, a

ph otomultiplier, an optical shutter, and at least one optical filter; and

00 ascanningunitcapableofexposingsaiddiscreteloca.onstohght

from said light source;

controlled by said computerized control mechanic.

28 Tto system of claim 26,w»erein sold grid holder is constructed of a,
..as, one mamri. selected from ma group ensisring of Unite, plastic, glass, siheon
and metal.

29. The system of claim 26, further comprising at least one robotic
mechanism.

30 The system of claim 29, wherein sand a, leas, ona robotic mechanism is
designed mad configured*, perfomring a, leas, one fnncrion seloced from mo *oup
consisting of:

(i) placing the grid holder into said loading device;

(ii) removing the grid holder fiom said loading device

(iii) transferring the grid holder to a scanning assay device;

(iv) removing the grid holder from said scanning assay device.

31 The system of claim 29, wherein said robotic mechanism includes at
oneconveyorbelt,atleasto„e P neumatictube,atleastonepistonandat,eastone
rotating plate.

PCT/US02/02660

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35

32 The system of claim 26, wherein said port comprises a first port

removal of said liquid from said space.

33 T hesystemofclaim26, .herein said electro-optical scanner further
comprises a cell manipulation device selected from the group consisting of a

device.

34 te sysM. of claim 33, *ta*> S"d ntaopipon. is capable of an

K ,ec,«d from .he g-oop conning of removing a. ieas. « portion of »
^.fromanindivi^c.^ovinga.ioartaportionofmem.v.dna.cogs

and moving nao individna, 0.1 from one of .aid macro* loe.oon,

,5 The system of olaim 33, wherein said needle la capable of an action

se ,ec,ad ftona me gronp co.sia.ing of injecting a auhsianco into an individual ce
^mngina-d^.iocaUo.^exnnonngaa.h^nomann^vadna.cen

residing in said discrete location.

36 The system of claim 33, wherein said electrode is capable of an action

of an individual cell residing in said discrete location, and creatrng a potential

37 Thesystemof claim 26, wherein at least one reagent contained within
said atlea.tone^^

the cells in the suspension at at least one wavelength.

PCT/US02/02660

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38 The system of claim 37, herein said at least one reagent capable of

a substance that differentially stains living cells;
apr ecursorofafluorescent substance tbat differentially stams hv.ng

(a)
(b)
cells;

(C ) a fluorophore that stains nucleic acids; and
(d) a flourescently labeled antibody.

39 The _ of Cairn 26, Ttomin said *«M*tai -~ ° f

pertaining to said photons.

m . Th es y9OT ofc,» m 39,»h«rei„»dp« 1 »«c,nd«a,s» S e f u.in
making a medical diagnosis.

„ An.n.on>..ed«>e.hodofe. n eeUc.nofda,. ft o m aplnra.iOof
mdividua. cells belonging . a popula-ion of c* in suspension, ft. method

"*T* , ^.----»'--«'»-»' f,-,, * ,- . <i r'

ed to » », snch to. each of said individoal disc«,e locahons .s

'"To^C^

^"dLochto.a.ow.c^eoftog.disinoommooicnhon^a

^T^---^»-"- ,, -' ,,, ' i ' , ~ ,,,,, ' 4 "' i

holder via at least one port; suspension
W causingtheindividualcellsfromthepopulataonofcellsms p

t0 Jintotheindividual discrete locations by means of a vacuum source

connectable to said port;

(d) supplying the population of cells in suspension;

PCT/US02/02660

WO 02/063034

„ M — — i - £~ - * - li,uid in at " °°°

^ «Li. mt iBaWidua. CDS from the pop»>.»on * — ™

ft joying «.o»d to6 device .ofacilte..—

susoension, and said at least one liquid reservoir,

^ 8) m^Oraindividna.ceUaraaidins^dind.v,^^

cardinal actions of said ce» earHa, rfd bolder, said vacoom

source, me r r f comput enzed control

loading device and said electro-opucal scanner by means

mechanism.

42 . The method of claim 41, wherein said *C*WP*- scanne,
""t .ropdcairrrnr.a.idopric.inni.coropHsi.^eantar.aUshts.nrce.a

controlled by said computerized control mechamsm.

43 Tbemethodofclaim41 ) whereinsaidgridholderisconstructedofat

least one materia! ^^^^^^^^
and metal.

PCT/US02/02660

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44 ^me^oM-imaf,— * —
least one robotic mechanism.

^ „ ,east one fonction selected from .he gmoP consisling of:

» removing said grid hold., from said loading dev.ce
i transferring said grid holder .o . scanning assay *«
„ ^ovingseidgridholderfsonrsaidscenningessaydevee.

46 The method of claim «, wherein said roho.icmeeh.nism includes a.

T realm fc gronp consisring - - «- - "** "
least on. item selected from the gro p ^

one conveyor belt, a. .««. one pnenma« "he, « least one p.s.on
rotating plate.

„ Theme.hodofc.aim4,, wherein said a, .east one port comprises a
^J^^onof.^----.^-—
for removal of said liqnid Horn said space.

48 •*»»«'■ comprise, the additional step of inclnding

device.

4Q Them ethodofclaim48 )W hereinsaidmicropipetteiscapableof

jrrstepselectedfromme^pconsistingofremovingatieasta

P6rf °^r I n olan individual ceH, removing at ^east a portion of the
portion of an organelle irom an in

Individual cell's cytoplasm, and removing the mdmdual cell from
locations.

PCT/XJS02/02660

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50. lnemcuiuu . ti „ su bstance into an

, ^ from the group consisting of injecting a suos

^dividual cell residing in sold disc*. loeaM.

— — — -- —

said discrete location.

52 ^mednodof e to 4 1> whe t «i„a,.e3 St o»er« g «n t eo„«i»«d

of:

(i)

cells; . ,

00 a fluorophore that stains nucleic acids; and
(iv) a flourescently labeled antibody.

portion of photons emanating from the ^ g ^ ^

discrete locations further includes gathenng polarization

photons.

PCTAJS02/02660

WO 02/063034

making a medical diagnosis.

^ » ,eas, .« po« for h— — * • - - *"*
liquid from said space;
M a vacuum source collectable to said port;

said at least one liquid reservoir; ^ ^ ^

whereinapplicauonof—

wherein said at least one liquid may be applied to the

wherein m surface of the

location selected from the group consistmg of said space an

cell carrier grid. illuminating the individual cells

m an electro-optical scanner capable of illuminating
(l)aneieiA " ^ii pr tina at least a portion of

„ i„ .aid individual discrete locations and collecting v

residmginsaidmmvi resi ding in said individual discrete

photons emanating from the individual cells residing

locations; and

PCT/US02/02660

WO 02/063034

■ 1 1 caniar grid ho.**, »d vacuum source, the popuhmon of cell. •»

suspension, said at least oh 4 with a graphical user

optical scanner, said computerized control mechanism operable with gr

interface.

57 me artsole of m— of chum 56, fur,.*, comprising motions

calibration thereof.

58 . The article of manufacture of claim 56, further comprising a cell
manipulation device.

^^.^ofm^c.usm.umgmp^.^.oca.ou.u.^

pWomul.lp.ier, . optica, shutter, „d e, l_ 0»e option! ^er

M aecllcame.grid.seidgridcempns.ngau.n.yofd.serct.

di^c.oca.ionaoopohleof engaging
W ascanmngunitcapab.e.f ..posing said diaom,. looahons ,o Ugh.

• o needle and an electrode and

m * cre T

co „ f ,gldfo,co—

seaming unit and said cell manipulation dev.no.

6 „ The elecUO-op.lc.l scanner of claim 59, wherein said micropipet* , ia
* rfon sa.eo.ad from «. gxoup consisting of removing aUaas. a porimn

0 ::Xt:o„drem OTi n 6 ,aindWidn.,uc,.uom„nanfsniud, S o re ,o,oo. 0 on S .

PCT/US02/02660

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42

61 Tneelec^opt^^^

' lected from the group consisting of injecting a substance mto an
of an action selected from me gr v cvtrnot in a a substance from an

individual cell residing in said discrete location.

fi9 The electro-optical scanner of claim 59, .herein said electrode is

lected from the group consisting of applying an electnc current
capable of an actum selected from gr P &

location.

-^■--^ —

(c) generating u ^„*+ pr and at least one
a camera, said light source, a photomultiplier, an opt.cal shutter, and

OPtiC T'' .-^I^^^^*^"*.,,. a

(d) mamp 8 tP afromthegroupconsistingofamicropipette,a

with a cell manipulation dev.ce selected from the gro p

needle, and an electrode; and tira , un it said cell carrier grid and .

saidcellmanipulationdeviceandsaidscannmgumt fromacontr
unit comprising a computer.

PCT/US02/02660

WO 02/063034

M Thexnethodofclain.eS.Xeinsaidmicropipetteiscapableof

64. The metnoa of removing at least

aportion of an organelle from he mdna ^ rf ^ ^

individual cell's cytoplasm, and removing the mdrv,

locations.

65 . l ne mem ; n ; PC tine a substance into an

individual cell residing in said discrete loca.cn and extractmg
individual cell residing in said discrete location.

66 The method of claim 63, wherein said electrode is capable

Mtional step selected from the group consisting of applymg an

rr:rr.r=r

individual cell residing in said discrete location.

WO 02/063034

1/9

PCT/US02/02660

I

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WO 02/063034

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PCT/US02/02660

WO 02/063034

3/9

PCT/US02/02660

WO 02/063034

4/9

PCT/US02/02660

WO 02/063034

5/9

PCT/XJS02/02660

and S_i

Place grid in holder.
(83)

imparting a measurable degree
of fluorescence (9 4)

r

-1 Contacting cells with j
1 liquid (9 0) ]

Remove from.scanning
assay device (101)

Remove grid holder.
(91)

Transfer, to scanning
assay device (92)

Manipulation of
individual cells (9 6)

illuminating and
collecting photons (9 5)

Removing an

individual cell or.

portion thereof (97)

Application of electrode to
cell (99)

Using a needle to inject
or extract (9 8)

WO 02/063034

6/9

PCT/DS02/02660

Figure 6c

WO 02/063034

7/9

PCT/US02/02660

Figure 6f

5

WO 02/063034

8/9

PCT/XJS02/02660

\ • w

PCT/X3S02/02660

WO 02/063034 9/9

> * *

INTERNATIONAL SEARCH REPORT

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f^^^b_* coated toing <1«

; 1 US 4,729,949 A l wcu 1093V see enure

„ pigHTON et al.) 16 November 1993 (16.ll.WW).
US5,262,128A(LElGHTONei __, entire document,

document. et „) 05 March 1996 (05.03.1996). see entrr

I A(PAR ' „ , WD ecemher 1999 -enure

wo 99 ,63049 Al (BIENERT et al.) 09 ^

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___—-. , r nn_WiUul -U«t

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considered to Involve an dements , «uch

to __«t member of the P-tcn. fanily

^uthorized^fficer
WUHam H. Beisnet
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.port

jean Proctor JUT
Paralegal s,^|/ ■ ■'

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